Color vision, which differentiates spectral compositions independent of brightness, provides animals, from insects to primates, great power for object recognition and memory registration and retrieval. Using a combination of genetic, histological, electrophysiological and behavioral approaches, we study how the visual system processes chromatic information to generate color percept in Drosophila. Using color preference and color learning assays, we demonstrated that flies innately prefer short wavelengths of light but they can be trained to select specific wavelengths of light by Pavlovian conditioning, indicating that flies, like honeybee and human, have true color vision. Using both light and electron microscope, we determined the synaptic circuits of the photoreceptors and their synaptic target neurons in the medulla ganglion of the peripheral visual system. We focused on the amacrine neurons, which interconnect medulla columns and the medulla projection (Tm) neurons, which are thought to serve functions analogous to those of vertebrate retinal ganglion cells by processing and relaying photoreceptor information to higher visual center. We found that the chromatic photoreceptors, R7 (UV-sensing) and R8 (blue/green-sensing), provide inputs to a subset of first-order interneurons, which might serve as color opponent neurons. The first-order interneurons Tm5a/b receive direct synaptic inputs from R7s while Tm9, Tm20 and Tm5c receive inputs from R8s. In addition, these Tm neurons receive indirect inputs from R1-6 via L3. These Tm neurons relay spectral information from the medulla to the higher visual center, the lobula. In addition to the direct pathways from photoreceptors to Tm neurons, the amacrine neuron Dm8 receives input from multiple R7s and provides input for Tm5a/b/c. To relate neural connectivity to functions, it is critical to assign components of synaptic machinery to specific connections. To directly probe the usage of neurotransmitters and receptors which determine the polarity and dynamic of signal transmission, we developed a method to profile transcripts in single neurons. We used highly specific promoter-Gal4 constructs to label single types of neurons with GFP and isolated these GFP-labeled neurons from adult fly brains and profiled their gene expression patterns by RT-PCR. Using this method, we determined that the majority of the first/second-order interneurons in the chromatic circuits express the vesicular glutamate transporter and the Kainate-type of ionotropic glutamate receptors (iGluR), indicating that they provide and receive fast, sign-conserving glutamate inputs in addition to their receiving of sign-inverting histaminergic inputs from photoreceptors. Our previous studies revealed that the amacrine Dm8 neurons are both required and sufficient for animals' innate spectral preference to UV light while Tm9 neurons are sufficient to drive green phototaxis. RNAi-knock-down of vesicular glutamate transporter in the Dm8 neurons significantly reduced UV preference, suggesting that glutamatergic output of Dm8 is critical for its functions. While Dm8 provides inputs for three types of neurons, Tm5a/b/c, inactivating Tm5c alone abolished UV preference, indicating that Tm5c is the key downstream targets for this behavior. Furthermore, RNAi-knockdonw of Kainate-type iGluR in Tm5c, thus inhibiting its ability to receive glutamatergic Dm8 inputs, significantly reduced UV preference. Using a modified GRASP (GFP-reconstitution across synaptic partners) method, which allows single-cell analysis of bona fide synaptic connections, we demonstrated that Dm8 receives 16 R7 inputs and provides inputs for 1-2 Tm5c neurons in the center of Dm8's receptive field. Together, we demonstrated that the R7s->Dm8->Tm5c connections constitute a hard-wired pooling circuit for detecting dim UV light.